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PubMed1989

Identification of Staphylococcus epidermidis vascular graft infections: a comparison of culture techniques.

Culture of prosthetic material is routinely used to exclude or implicate infection in the pathogenesis of late-appearing graft complications. In a canine model of aortic graft infection caused by a bacterial biofilm, the influence of culture media (blood agar and tryptic soy broth) and mechanical surface biofilm disruption (tissue grinding and ultrasonic oscillation) on microorganism recovery was determined. Dacron prostheses colonized in vitro with Staphylococcus epidermidis were implanted in the infrarenal aortas of 36 dogs. After 3 weeks an infection with anatomic characteristics of late graft infection in humans was present. Explantation (+/- surface biofilm disruption) of infected grafts showed broth culture was superior (p less than 0.001) to agar media in confirming infection. The recovery rate of S. epidermidis was 30% with agar media, was 72% with broth media alone, and was 83% with broth media plus biofilm disruption. In situ replacement of infected grafts plus parenteral antibiotics resulted in early (1 month) healing of 31 grafts without signs of infection. All replacement grafts were sterile when cultured in broth media alone, but the addition of biofilm disruption isolated the study strain from eight (22%) of 36 grafts (p less than 0.01). Biofilm disruption by tissue grinding or sonication increased bacteria recovery equally. When biofilm bacterial concentration was less than 100 colony-forming units/cm2 of graft, only culture in broth media reliably recovered microorganisms. In the absence of perigraft inflammation, microbiologic recovery techniques that identify bacterial biofilms are necessary to exclude infection in studies concerning the pathogenesis of late graft complications or the treatment of S. epidermidis prosthetic infections.

Animals↗

PubMed1991

Biocompatibility evaluation of glass ionomer cement using cell culture techniques.

Cell culture techniques have been employed to carry out a preliminary investigation into the biocompatibility of a glass ionomer cement (GIC). A modification of the commonly used Agar Overlay test and a rapid, colorimetric assay based on the reduction of tetrazolium salt were used. The GIC is shown to leach a cytotoxic agent, possibly fluoride. This agent is effectively removed by an extraction procedure, and re-examination of the GIC demonstrates its cytocompatibility.

Agar↗

PubMed1963

WHO CO-OPERATIVE STUDIES ON A SIMPLE CULTURE TECHNIQUE FOR THE ISOLATION OF MYCOBACTERIA. 1. PREPARATION, LYOPHILIZATION AND RECONSTITUTION OF A SIMPLE SEMI-SYNTHETIC CONCENTRATED LIQUID MEDIUM; CULTURE TECHNIQUE; GROWTH PATTERN OF DIFFERENT MYCOBACTERIA.

Tuberculosis surveys are in progress in many countries that do not have adequate laboratory facilities for carrying out complicated bacteriological procedures. As part of a WHO co-operative research programme, studies have been undertaken with a view to developing a simple culture technique for the isolation of mycobacteria that does not require elaborate equipment. This paper is the first report on these co-operative studies.Storage and transport are known to affect adversely the viability of Mycobacterium tuberculosis in pathological specimens, thus giving rise to poor culture results and indicating the advisability of culturing such specimens on the spot. The preparation of the efficient and widely used Löwenstein-Jensen (L-J) culture medium, however, requires materials and facilities that are not easy available in developing countries. In an attempt to overcome this difficulty, the Tuberculosis Research Institute in Prague has developed a semi-synthetic liquid medium that can be prepared in bulk, concentrated and lyophilized, and sent even to distant laboratories.The present paper describes in detail the preparation of this lyophilized medium, which can be stored at room temperature for at least 6-12 months and is easy to reconstitute, and discusses the growth characteristics of mycobacteria multiplied in it.Experience in Czechoslovakia, where between 1953 and 1962 nearly 21 million cultures have been made with the medium, has shown that it is quite satisfactory and even slightly superior to L-J medium in certain respects.

Bacteriological Techniques↗

PubMed1975

Practical aerobic membrane filtration blood culture technique: clinical blood culture trial.

During the course of preliminary clinical trials of an improved membrane filter blood culture system, filter plugging produced by a gelling of the lysing solution was observed when the patients had high leukocyte counts. A solution of streptokinase-streptodornase (Varidase, Lederle Laboratories) dissolved or prevented the gel and permitted rapid filtration without plugging. With streptokinase-streptodornase incorporated in the filtration procedure, a comparison of several culture systems was carried out on 176 blood cultures. Brucella broth with and without sodium polyanethol sulfonate, a prereduced osmotically stabilized broth, pour plates, and an improved aerobic membrane filter system were compared. The membrane filter system yielded 29 of the total of 37 positive cultures, far surpassing all other systems. Eight of these cultures were detected first by the filter technique, and 13 were positive only in this system. Nineteen of the 37 positive cultures were from patients on antimicrobial agents. Fourteen of these were detected by the filter, twice the number detected by any of the other systems.

Aerobiosis↗

PubMed1994

[Advances in culture techniques for airway epithelial cells].

Techniques for culturing the airway epithelium have been improved over the past 10 years. Cultured cells show multilayered structures, and the luminal surface contains cilia and secretory granules. The cells retain the ion transport properties of the original tissue, and secrete mucin-like glycoproteins indistinguishable from mucins produced in vivo. The culture conditions were improved in several ways. A serum-free hormone-supplemented culture medium, growth supports such as collagen or collagen gel, and filter membranes or culture dishes suitable for epithelial cell culture are now used, and air interface methods have been developed. Furthermore, protease can be used to harvest many cells from the airway epithelium. This paper introduces the history of the culture techniques for the airway epithelium over the past 10 years, and recent improvements in the culture conditions for human tracheal epithelium and submuiosal glands.

Animals↗

PubMed2003

Transplantation of cultured bone cells using combinations of scaffolds and culture techniques.

The transplantation of cultured bone cells is expected to become a candidate for bone regeneration therapy. For the clinical application of this therapy, there remain several problems to be overcome, for example, the improvements of scaffolds and culture techniques. In this review article, two kinds of porous ceramics, a novel sintered porous hydroxyapatite and a porous beta-tricalcium phosphate (TCP), as well as a collagen-phosphosphoryn sponge are introduced as new scaffolds for bone regeneration. The former two ceramic scaffolds proved to be applicable for bone regeneration therapy. The collagen-phosphophoryn sponge proved to have bone formation ability in vivo. Moreover, for the application of this therapy to the regeneration of large bone defects, we improved the culture method by applying a low-pressure system and a perfusion system. Both culture systems accelerated the formation of bone in vivo in this transplantation model. Combinations of the scaffolds and culture techniques might be considered when designing therapeutic strategies.

Animals↗

PubMed1987

Effect of different levels of phosphorus on rumen microbial fermentation and synthesis determined using a continuous culture technique.

A continuous culture technique was used to study the phosphorus requirements of rumen micro-organisms. Solutions of artificial saliva containing 120, 80, 40 and 0 mg inorganic phosphorus (Pi)/l were infused into the reaction vessels previously inoculated with rumen contents, resulting in Pi concentrations in the vessel contents of 48, 28, 4 and less than 1 mg/l respectively. Various fermentative and synthetic characteristics were examined. In the vessel contents, concentrations of protozoa (about 0.9 X 10(5)/ml) were not significantly affected by Pi concentration. Total volatile fatty acids (VFA) produced averaged about 6.83 mmol/h with Pi levels of 48 and 28 mg/l. Reduction in Pi concentrations to 4 and less than 1 mg/l resulted in significant reductions in total VFA to approximately 6.25 and 3.75 mmol/h respectively, accompanied by a rise in pH from 6.5 to 7.3. Ammonia-nitrogen values, which averaged about 131 mg/l at the higher Pi concentrations, also increased with the lowest level of Pi to about 240 mg/l. ATP concentrations averaged about 14 mumol/l at the highest Pi concentration and fell progressively with each reduction in Pi concentration to a final value of 2.5 mumol/l with the Pi level less than 1 mg/l. At Pi concentrations of 48 and 28 mg/l, the digestibilities of xylose, arabinose and cellulose-glucose were maintained at about 0.90, 0.62 and 0.70 g/g input respectively. At lower Pi concentrations these digestibilities fell significantly and corresponding values at Pi less than 1 mg/l were 0.73, 0.41 and 0.31 respectively. Starch digestion was unaffected by Pi concentration and remained at about 0.90 g/g input. The amount of microbial-N synthesized averaged 0.48 g/d and was maintained with Pi concentrations down to 4 mg/l. There was, however, a significant reduction to 0.26 g/d with Pi concentrations of less than 1 mg/l. The efficiency of microbial protein synthesis was variable but averaged approximately 25 g N/kg total carbohydrate fermented. It was estimated that the minimum Pi concentrations required in rumen fluid in vivo to maintain maximum degradative and synthetic microbial activities was in the range 75-100 mg/l and that over-all P requirement of the microbes was of the order of 5.1 g/kg apparently digested organic matter intake.

Animal Nutritional Physiological Phenomena↗

PubMed1979

Generation time statistics of Escherichia coli B measured by synchronous culture techniques.

Synchronous cultures of Escherichia coli B were produced under various environmental conditions. Analysis of the cell number data permitted the characterization of the generation time distribution for these organisms and the estimation of the mother-daughter generation time correlation coefficients. For all growth conditions, the distribution of generation times was found to be symmetrical with a coefficienoefficient was significantly negative at doubling times between 40 and 64 min. However, the results for a culture growing in succinate medium at 37 degrees C, which had a significantly greater generation time, yielded a correlation coefficient close to zero. Within the range of temperatures studied (26 to 37 degrees C), no significant effect on the correlation coefficient was observed.

Bacteriological Techniques↗

PubMed1980

Studies of renal cell function using cell culture techniques.

Cell culture, a powerful tool for the study of cell biology, offers advantages for the study of renal cell function. Epithelial cells derived from a variety of organs, including the kidney, form oriented epithelial sheets in culture that have many structural characteristics (microvilli, tight junctions) of epithelia in situ. There is evidence of transepithelial transport of salt and water by cells of two lines (MDCK and LLC-PK1) derived from mammalian kidney. LLC-PK1 cells may also manifest the glucose transport system of the proximal tubule. Cells of both lines have adenylate cyclase activity sensitive to hormones. Two lines of cells derived from toad urinary bladder form epithelia with a high transepithelial resistance and transport sodium actively from apical to basolateral surface. The rate of sodium transport in both lines is stimulated by cyclic AMP and by aldosterone. There are important differences in the characteristics of the response of the two lines to aldosterone as well as in their sensitivity to inhibition of sodium transport by amiloride. These differences may lead to new insights regarding the molecular events in the response to aldosterone and in the inhibitory action of amiloride. Cultures of kidney cells have also been used effectively to study the biosynthesis of the hormonal derivative of vitamin D and to study prostaglandin production. In addition, cell culture is ideally suited for study of the developmental biology of the kidney.

Aldosterone↗

PubMed1977

Development of a lysis-filtration blood culture technique.

A lysed-blood culture system that quickly lyses patients' blood near neutrality and is relatively noninjurious to more delicate pathogens such as Haemophilus influenzae and Bacteroides fragilis is reported. The lysing solution includes culture medium, 0.004 M sodium carbonate and bicarbonate, 0.04% Triton X-100,and 0.6% Rhozyme (a mixture of proteases). Most of the pathogens tested multiplied in the lysing solution. The lysed blood normally is immediately filtered. The membrane is transferred to culture broth. The greatest advantage realized from this blood culture technique is separation of pathogens from antibiotics, bactericidal antibodies, complement, opsonins, and phagocytic systems. Another advantage is the concentration of organisms into a small volume of clear medium for faster growth and visualization of growth. It was observed that both gram-negative and -positive organisms were attracted during filtration to the filter material and were not removed from it by backwashing with buffer. Thus, filter membranes with porosities much larger than would nominally be expected to retain bacteria retained all or part of light and heavy Escherichia coli and Staphylococcus aureus suspensions. Advantage may be taken of this phenomenon to use filters with larger pore sizes and avoid filter clogging by poorly lysed specimens. Porr lysis may result from addition of too much blood to the lysing solution, blood with elevated numbers of erythrocytes or leukocytes, or blood from some people whose blood is naturally more resistant to lysis.

Bacteriological Techniques↗

PubMed2005

Detection of Kluyveromyces marxianus and other spoilage yeasts in yoghurt using a PCR-culture technique.

A combined PCR-culture technique was developed for the detection of viable yeasts in yoghurt samples. Yoghurt samples were inoculated with either viable or heat-inactivated Kluyveromyces marxianus cells, and analyzed before and after incubation for 24 h at 25 degrees C under agitation. DNA was extracted from the samples and amplified using yeast-specific primers targeted at the gene coding for the 18S rRNA. A 251-bp fragment was amplified by the Polymerase Chain Reaction from the yoghurt samples containing initial yeasts counts of 10 cfu g(-1) or higher, whereas no PCR product was generated from control uninoculated yoghurt samples. Comparison of PCR results obtained before and after the incubation step was used to assess yeast viability. Viability was also confirmed by plating on Sabouraud-Dextrose-Chloramphenicol Agar. Moreover, comparison of the results obtained using PCR-culture and plate count methods for the analysis of commercial yoghurt samples, demonstrated that the PCR-culture technique developed in this work can be very useful for the rapid detection of viable spoilage yeasts in dairy industries.

Colony Count, Microbial↗

PubMed1964